Funding support from the National Natural Science Foundation of China (grant 31870941), Natural Science Foundation of Shanghai (grant 22ZR1429600), and the Science and Technology Commission of Shanghai Municipality (grant 19441907700) is acknowledged.

1. Gelatin phantom preparation
<ol>
	<li>Weigh gelatin, glycerol, and water according to Table 1. Mix the gelatin powder with water to obtain the gelatin solution. 
	NOTE: The concentrations of the individual components for preparing the two phantoms are shown in Table 1. The higher the concentration of gelatin, the stiffer the phantom.</li>
	<li>Heat the gelatin solution to 60 &#176;C in a water bath. Add glycerol to the gelatin solution while maintaining the temperature. 
	NOTE: Glycerol stabilizes gelatin mixtures by increasing their melting temperature and shear modulus17.</li>
	<li>Stir the solution and heat it to 60 &#176;C again. Pour the mixed solution into a container that will be used for MRE and indentation tests. Cool the solution to room temperature and wait till the solution is solidified.</li>
</ol>
2. MRE test
<ol>
	<li>Put the vibration plate on top of the gelatin phantom. Ensure that the contact between the phantom and the vibration plate is firm (Figure 1A). 
	NOTE: The vibration plate is made of Polyamide with a dimension of 50 x 50 x 5 mm3.</li>
	<li>Place the gelatin phantom into the head coil. Put sponges and sandbags around the gelatin phantom to make sure the phantom is firmly placed. Use a custom-built electromagnetic actuator with a transmission bar14,18. Mount an electromagnetic actuator on the head coil. Connect the transmission bar to the vibration plate (Figure 1B).</li>
	<li>Connect the power lines of the actuator with the amplifier. Connect the control lines with the controller (Figure 1C).</li>
	<li>Actuator and MRI scan parameter settings
	<ol>
		<li>Set the waveform, vibration frequency, and amplitude in the function generator. Set the desired vibration amplitude by adjusting the power amplifier. 
		NOTE: Here, the waveform is set to sinusoidal in the function generator; the vibration frequency is set to 40 Hz or 50 Hz, and the amplitude is set to 1.5 Vpp. In the power amplifier, the amplification ratio is set to 40%.</li>
		<li>Set the function generator to work in the trigger mode. Connect the trigger line to the external trigger port of the MRI machine.</li>
		<li>Set the MRE scanning (actuator) frequency the same as that from the function generator, so that the motion encoding gradient is synchronized with the motion of the vibration plate.</li>
	</ol>
	</li>
	<li>Data measurement and analysis
	<ol>
		<li>Follow the routine imaging positioning procedures. Use a 2D gradient-echo (GRE) based MRE sequence for imaging of the gelatin phantom20. Set the GRE-MRE imaging parameters as follows: Flip-angle = 30&#176;; TR/TE = 50/31 ms; Field-of-view = 300 mm; Slice thickness = 5 mm; Voxel size = 2.34 x 2.34 mm2.</li>
		<li>Measure the phase images at four temporal points in one sinusoidal cycle. Apply both positive and negative motion encoding gradients at each time point.</li>
		<li>Based on the phase image acquired, remove the background phase by subtracting the positive and negative encoded phase images. Unwrap the phase with a reliability sorting-based algorithm21.</li>
		<li>Extract the principal component of the motion by applying fast Fourier Transform to the unwrapped phase images. Filter the phase image with a digital bandpass filter. Estimate the shear modulus with a 2D direct inversion (DI) algorithm to obtain storage modulus G&#39; and loss modulus G&#39;&#39;13,14. 
		&#8203;NOTE: The cut-off frequency of the bandpass filter is [0.04 0.08]. The size of the fitting window of the DI algorithm is 11 x 11.</li>
	</ol>
	</li>
</ol>
3. Indentation test
<ol>
	<li>Use a circular punch or surgical blade to trim the gelatin phantom into a cylindrical or cuboid sample, respectively. Make sure that the sample thickness is between 3 and 10 mm and the diameter of the cylindrical sample or the long side of the cuboid is larger than 4 mm. Use a sharp blade to trim the surface of the sample to make it as smooth as possible for indentation.</li>
	<li>Turn on the power of the indentation tester. Perform the following using the indenter control program designed to automate the indenter contact procedure (custom program; see Table of Materials).
	<ol>
		<li>Click on the Back off button in the GUI to initialize the calibration process (Figure 2B). Read the value from the laser sensor and type the value in the BaseLine box. 
		NOTE: During the calibration process, the distance between the laser sensor and the baffle plate is adjusted to a specific pre-defined value.</li>
		<li>Place a glass slide on the baffle plate and record the value shown by the laser sensor. Next, put the sample on the glass slide and place them together on the baffle plate. Read the value from the laser sensor and type this value in the Sample+Slide box. 
		NOTE: The laser sensor is used to record the displacement of the indentation, but it is also used to measure the sample thickness before the test.</li>
		<li>Take the difference between the two values obtained in step 3.2.2 as the sample thickness at the region of interest (ROI).</li>
		<li>Carefully place the sample along with the underlying glass slide right below the indenter, and then click on the Contact button to initiate automatic contact between the indenter and the sample surface. 
		NOTE: If the automatic contact is not satisfactory, i.e., the indenter presses deep into the sample or does not have a contact, adjust the indenter position by typing a value in the range of 0.05-0.1 mm in the Offset box and repeat steps 1.2.1-1.2.4.</li>
		<li>Based on the measured sample thickness (step 3.2.3), estimate the indentation displacement (i.e., total indentation depth) by multiplying the thickness with the indented testing strain (here, it is set to &#8804;8% to keep the indentation within the small strain assumption).</li>
		<li>Type the displacement values (step 3.2.5) in the Displacement (mm) box. Set the relaxation time to 180 s in the Dwell Time box. Click on the Indentation button. The displacement and reactive force during the ramp-hold procedure will be automatically recorded and saved in a file at the specified File Path. 
		NOTE: The File Path can be pre-defined as the path for saving testing data.</li>
	</ol>
	</li>
	<li>Export the indentation data to a spreadsheet. Use a two-term Prony series <img alt="Equation 1" src="/files/ftp_upload/63770/63770eq01.jpg" style="margin: auto;" /> to fit the force relaxation curve1,3,11: 
	<img alt="Equation 2" src="/files/ftp_upload/63770/63770eq02.jpg" style="margin: auto;" /></li>
	<li>Estimate the instantaneous shear modulus (G0) and long-term shear modulus (G&#8734;) based on the fitted parameters: 
	<img alt="Equation 3" src="/files/ftp_upload/63770/63770eq03.jpg" style="margin: auto;" /> 
	NOTE: In the above equations, C0, Ci, and &#964;i are model parameters of the Prony series, F is the indentation force, R is the radius of the indenter, X is the compensation factor for the infinite half space assumption, V is the indentation velocity, t is the time variable, and tR is the ramp time.</li>
</ol>

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Authors declare no conflicts of interest.

Gelatin phantoms are commonly used as tissue-mimicking materials for testing and validation of algorithms and devices17,19,22,23,24,25,26,27. One of the pioneering studies using the gelatin phantom to compare MRE and dynamic shear testing was presented by Okamoto et al. (2011)17. In their study, the mass concentration of the gelatin was ~2.8%, and the estimated G&#39; and G&#39;&#39; values after correction were in the ranges of 1.06-1.15 kPa and 0.11-0.27 kPa, respectively. Zeng et al. (2020)19 also used gelatin phantom to validate the inversion algorithm for MRE. With a gelatin mass concentration of ~3.5%, the estimated G&#39; value was ~2.5 kPa. Since the shear modulus increases with the concentration of gelatin, these values were all consistent with the estimation in this study.
To make gelatin phantoms, it is noted that a complete and thorough stirring is required when mixing a large amount of gelatin powder with water. This is necessary for full dissolution to make homogenized phantoms. To increase the melting temperature and shear modulus, glycerol was added to the mixture17. The water bath at around 60 &#176;C will help accelerate the mixing and is recommended during the stirring process. Usually, the gelatin will be formed in a container with a specific shape, e.g., cube or cylinder. Therefore, it is important to filter out the bubbles before pouring the mixed solution into the container.
When preparing for the MRE test, a stable transmission of the shear wave is crucial. Therefore, it is necessary to make sure the vibrating plate is firmly pressed on top of the phantom. This is to avoid any possible slipping between the plate and the phantom. However, this will potentially bring a certain level of local pre-stress. Thus, it is important not to over-press the plate on the phantom. When setting up the actuation frequency, it is noted that the damping of the wave propagation increases with the frequency.&#160;
It is suggested to place the indentation device on a vibration isolation table. This is because even a small vibration will affect the ramp-hold recording process. In addition, re-calibration of the sensors is needed if the device has not been used for more than 1 month.
To have the best measurement performance of MRE, it is suggested to keep the frequency within 100 Hz. This is because the higher the frequency, the more dissipation of the vibration, thus inducing a lower SNR of the images acquired. The indentation test mainly measures the sample at a frequency range lower than that of MRE. For a discussion of the parameter conversions between the two methods, one can refer to Chen et al. (2020)11. The MRE and indentation can be used to measure many soft biological tissues to investigate the biomechanical properties and explore the potential biomechanics-based biomarkers for disease diagnosis or treatment evaluation.

Most of the soft biological tissues appear to have viscoelastic properties that are important to understand their injury and development1,2. In addition, viscoelastic properties are important biomarkers in the diagnosis of a variety of diseases such as fibrosis and cancer3,4,5,6. Therefore, the characterization of viscoelastic properties of soft tissues is crucial. Among the many characterization techniques used, ex vivo mechanical testing of tissue samples and in vivo elastography using biomedical imaging are the two widely used methods.
Although various mechanical testing techniques have been used for soft tissue characterization, the requirements for sample size and testing conditions are not easy to be satisfied. For example, shear testing needs to have samples fixed firmly between the shear plates7. Biaxial testing is more suitable for membrane tissue and has specific clamping requirements8,9. A compression test is commonly used for tissue testing, but cannot characterize specific positions within one sample10. The indentation test does not have additional requirements to fix the tissue sample and can be used to measure many biological tissue samples such as the brain and liver. In addition, with a small indenter head, regional properties within a sample could be tested. Therefore, indentation tests have been adopted to test a variety of soft tissues1,3,11.
Characterizing the biomechanical properties of soft tissues in vivo is important for translational studies and clinical applications of biomechanics. Biomedical imaging modalities such as ultrasound (US) and magnetic resonance (MR) imaging are the most used techniques. Although US imaging is relatively cheap and easy to carry out, it suffers from low contrast and is hard to measure organs such as the brain. Capable of imaging deep structures, MR Elastography (MRE) could measure a variety of soft tissues6,12, especially the brain13,14. With applied external vibration, MRE could measure the viscoelastic properties of soft tissues at a specific frequency.
Studies have shown that at 50-60 Hz, the shear modulus of the normal brain is ~1.5-2.5kPa5,6,13,14,15 and ~2-2.5 kPa for normal liver16. Therefore, gelatin phantoms that have similar biomechanical properties have been widely used for mimicking soft tissues for testing and validation17,18,19. In this protocol, gelatin phantoms with two different concentrations were prepared and tested. Viscoelastic properties of the gelatin phantoms were characterized using a custom-built electromagnetic MRE device14 and an indentation device1,3. The testing protocols could be used for testing many soft tissues such as the brain or liver.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-channel head &#38; Neck coil</td>
			<td>United Imaging Healthcare</td>
			<td>100120</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>3T MR Scanner</td>
			<td>United Imaging Healthcare</td>
			<td>uMR 790</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Acquisition board</td>
			<td>Advantech Co</td>
			<td>PCI-1706U</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Computer-Windows</td>
			<td>HP</td>
			<td>790-07</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Electromagnetic actuator</td>
			<td>Shanghai Jiao Tong University</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>RIGOL</td>
			<td>DG1022Z</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>CARTE D&#8217;OR</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Vance Bioenergy Sdn.Bhd</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Indenter control program</td>
			<td>custom-designed</td>
			<td></td>
			<td>Software; accessed via: https://github.com/aaronfeng369/FengLab_indentation_code.</td>
		</tr>
		<tr>
			<td>Laser sensor</td>
			<td>Panasonic</td>
			<td>HG-C1050</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>Transducer Technique</td>
			<td>GSO-10</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Power amplifier</td>
			<td>Yamaha</td>
			<td>A-S201</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Voice coil electric motor</td>
			<td>SMAC Corporation</td>
			<td>DB2583</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

viscoelastic characterization of soft tissue-mimicking gelatin phantoms using indentation and magnetic resonance elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

Following the MRE protocol, a clear shear wave propagation in the gelatin phantoms at 40 and 50 Hz were observed (Figure 3). The viscoelastic properties measured from MRE, and indentation tests are shown in Figure 4. The estimated G&#39; and G&#34; values at each testing for each phantom are summarized in Table 2. Following the indentation protocol, the viscoelastic properties of each phantom at each test point are summarized in Table 3.
As shown in Figure 4, for measurements using MRE, a comparison of G&#39; and G&#34; values measured at 40 and 50 Hz showed significant differences between the two gelatin phantoms (student&#39;s t-test, p &#60; 0.05). In addition, significant differences were observed for both G&#39; and G&#34; values between 40 and 50 Hz measurements (student&#39;s t-test, p &#60; 0.05). Similarly, for measurements using indentation test, significant differences between the two phantoms were observed for G0 and G&#8734; values (student&#39;s t-test, p &#60; 0.05). Both MRE and indentation provided consistent results for distinguishing soft and stiff gelatin phantoms.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63770/63770fig01.jpg" /> 
Figure 1: MRE test. (A) Put the vibration plate on top of the gelatin phantom. (B) Place the gelatin phantom inside the head coil and mount the electromagnetic actuator on top of the head coil. (C) An overview of the electromagnetic MRE system showing the connections between each component. <a href="https://www.jove.com/files/ftp_upload/63770/63770fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63770/63770fig02.jpg" /> 
Figure 2: Indentation test. (A) Put the gelatin phantom right under the indenter head in the tester. (B) Prepare the indentation using the Control Setup panel in the GUI. Input the indentation parameters in the GUI to set up the ramp-relaxation test. Observe the indentation curves in the Data Viewer window. <a href="https://www.jove.com/files/ftp_upload/63770/63770fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63770/63770fig03.jpg" /> 
Figure 3: Wave propagation images for the two gelatin phantoms at 40 and 50 Hz. The four phases correspond to the four temporal points at one sinusoidal cycle. <a href="https://www.jove.com/files/ftp_upload/63770/63770fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63770/63770fig04.jpg" /> 
Figure 4: Viscoelastic properties measured from MRE and indentationexperiments.&#160;(A) Typical estimated G&#39; and G&#39;&#39; maps at 40 and 50 Hz for the two gelatin phantoms from MRE. (B) Mean and standard deviation of the G0 and G&#8734; values for the two phantoms from six repeated indentation tests. (C) Mean and standard deviation of the G&#39; and G&#39;&#39; values at 40 and 50 Hz for the two phantoms from six repeated MRE tests. The asterisk symbol indicates a significant difference (student&#39;s t-test; p &#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/63770/63770fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Gelatin</td>
			<td>Water</td>
			<td>Glycerol</td>
			<td>Total</td>
		</tr>
		<tr>
			<td>Phantom 1</td>
			<td>100 (4.35%)</td>
			<td>1200 (52.17%)</td>
			<td>1000 (43.48%)</td>
			<td>2300 (100%)</td>
		</tr>
		<tr>
			<td>Phantom 2</td>
			<td>160 (6.96%)</td>
			<td>1140 (49.56%)</td>
			<td>1000 (43.48%)</td>
			<td>2300 (100%)</td>
		</tr>
	</tbody>
</table>
Table 1: The mass and mass concentration of the gelatin, glycerol, and water used for preparing the two gelatin phantoms. The mass unit is grams.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3" style="min-width: 80px;">Modulus(Pa)</td>
			<td style="min-width: 80px;">Test 1</td>
			<td style="min-width: 80px;">Test 2</td>
			<td style="min-width: 80px;">Test 3</td>
			<td style="min-width: 80px;">Test 4</td>
			<td style="min-width: 80px;">Test 5</td>
			<td style="min-width: 80px;">Test 6</td>
			<td style="min-width: 80px;">Mean</td>
			<td style="min-width: 80px;">Std</td>
		</tr>
		<tr>
			<td rowspan="4" style="min-width: 80px;">Phantom 1</td>
			<td rowspan="2" style="min-width: 80px;">40 Hz</td>
			<td style="min-width: 80px;">G&#39;</td>
			<td style="min-width: 80px;">2978</td>
			<td style="min-width: 80px;">2976</td>
			<td style="min-width: 80px;">2976</td>
			<td style="min-width: 80px;">2974</td>
			<td style="min-width: 80px;">2971</td>
			<td style="min-width: 80px;">2972</td>
			<td style="min-width: 80px;">2975</td>
			<td style="min-width: 80px;">3</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">G&#39;&#39;</td>
			<td style="min-width: 80px;">198</td>
			<td style="min-width: 80px;">197</td>
			<td style="min-width: 80px;">197</td>
			<td style="min-width: 80px;">198</td>
			<td style="min-width: 80px;">199</td>
			<td style="min-width: 80px;">199</td>
			<td style="min-width: 80px;">198</td>
			<td style="min-width: 80px;">1</td>
		</tr>
		<tr>
			<td rowspan="2" style="min-width: 80px;">50 Hz</td>
			<td style="min-width: 80px;">G&#39;</td>
			<td style="min-width: 80px;">2854</td>
			<td style="min-width: 80px;">2852</td>
			<td style="min-width: 80px;">2852</td>
			<td style="min-width: 80px;">2851</td>
			<td style="min-width: 80px;">2850</td>
			<td style="min-width: 80px;">2848</td>
			<td style="min-width: 80px;">2851</td>
			<td style="min-width: 80px;">2</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">G&#39;&#39;</td>
			<td style="min-width: 80px;">341</td>
			<td style="min-width: 80px;">342</td>
			<td style="min-width: 80px;">342</td>
			<td style="min-width: 80px;">342</td>
			<td style="min-width: 80px;">341</td>
			<td style="min-width: 80px;">341</td>
			<td style="min-width: 80px;">341</td>
			<td style="min-width: 80px;">1</td>
		</tr>
		<tr>
			<td rowspan="4" style="min-width: 80px;">Phantom 2</td>
			<td rowspan="2" style="min-width: 80px;">40 Hz</td>
			<td style="min-width: 80px;">G&#39;</td>
			<td style="min-width: 80px;">5603</td>
			<td style="min-width: 80px;">5589</td>
			<td style="min-width: 80px;">5596</td>
			<td style="min-width: 80px;">5590</td>
			<td style="min-width: 80px;">5586</td>
			<td style="min-width: 80px;">5588</td>
			<td style="min-width: 80px;">5592</td>
			<td style="min-width: 80px;">7</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">G&#39;&#39;</td>
			<td style="min-width: 80px;">419</td>
			<td style="min-width: 80px;">412</td>
			<td style="min-width: 80px;">419</td>
			<td style="min-width: 80px;">413</td>
			<td style="min-width: 80px;">408</td>
			<td style="min-width: 80px;">408</td>
			<td style="min-width: 80px;">413</td>
			<td style="min-width: 80px;">5</td>
		</tr>
		<tr>
			<td rowspan="2" style="min-width: 80px;">50 Hz</td>
			<td style="min-width: 80px;">G&#39;</td>
			<td style="min-width: 80px;">5343</td>
			<td style="min-width: 80px;">5341</td>
			<td style="min-width: 80px;">5336</td>
			<td style="min-width: 80px;">5336</td>
			<td style="min-width: 80px;">5329</td>
			<td style="min-width: 80px;">5331</td>
			<td style="min-width: 80px;">5336</td>
			<td style="min-width: 80px;">6</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">G&#39;&#39;</td>
			<td style="min-width: 80px;">317</td>
			<td style="min-width: 80px;">317</td>
			<td style="min-width: 80px;">318</td>
			<td style="min-width: 80px;">324</td>
			<td style="min-width: 80px;">321</td>
			<td style="min-width: 80px;">323</td>
			<td style="min-width: 80px;">320</td>
			<td style="min-width: 80px;">3</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Storage modulus (G&#39;) and loss modulus (G&#34;) of the two gelatin phantoms measured by MRE. Each phantom was tested six times at an actuation frequency of 40 and 50 Hz.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Test 1</td>
			<td>Test 2</td>
			<td>Test 3</td>
			<td>Test 4</td>
			<td>Test 5</td>
			<td>Test 6</td>
			<td>Mean</td>
			<td>Std</td>
		</tr>
		<tr>
			<td rowspan="8">Phantom 1</td>
			<td>C0</td>
			<td>1.54</td>
			<td>1.88</td>
			<td>1.81</td>
			<td>1.71</td>
			<td>1.65</td>
			<td>1.60</td>
			<td>1.70</td>
			<td>0.13</td>
		</tr>
		<tr>
			<td>C1</td>
			<td>0.64</td>
			<td>0.16</td>
			<td>0.09</td>
			<td>0.16</td>
			<td>0.16</td>
			<td>0.21</td>
			<td>0.23</td>
			<td>0.20</td>
		</tr>
		<tr>
			<td>C2</td>
			<td>0.10</td>
			<td>0.12</td>
			<td>0.15</td>
			<td>0.11</td>
			<td>0.13</td>
			<td>0.11</td>
			<td>0.12</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>&#964;1 (s)</td>
			<td>459.71</td>
			<td>177.52</td>
			<td>114.14</td>
			<td>7.32</td>
			<td>6.1</td>
			<td>3.73</td>
			<td>128.09</td>
			<td>177.51</td>
		</tr>
		<tr>
			<td>&#964;2 (s)</td>
			<td>9.83</td>
			<td>6.38</td>
			<td>5.83</td>
			<td>199.28</td>
			<td>200.2</td>
			<td>55.78</td>
			<td>79.55</td>
			<td>94.98</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>0.99</td>
			<td>1.00</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>G0 (Pa)</td>
			<td>2273</td>
			<td>2145</td>
			<td>2040</td>
			<td>1991</td>
			<td>1935</td>
			<td>1920</td>
			<td>2051</td>
			<td>136</td>
		</tr>
		<tr>
			<td>G&#8734; (Pa)</td>
			<td>1535</td>
			<td>1875</td>
			<td>1808</td>
			<td>1714</td>
			<td>1650</td>
			<td>1601</td>
			<td>1697</td>
			<td>128</td>
		</tr>
		<tr>
			<td rowspan="8">Phantom 2</td>
			<td>C0</td>
			<td>5.97</td>
			<td>6.29</td>
			<td>6.16</td>
			<td>6.20</td>
			<td>6.14</td>
			<td>6.11</td>
			<td>6.14</td>
			<td>0.11</td>
		</tr>
		<tr>
			<td>C1</td>
			<td>0.29</td>
			<td>0.30</td>
			<td>0.43</td>
			<td>0.38</td>
			<td>0.18</td>
			<td>0.48</td>
			<td>0.34</td>
			<td>0.11</td>
		</tr>
		<tr>
			<td>C2</td>
			<td>0.64</td>
			<td>0.24</td>
			<td>0.24</td>
			<td>0.17</td>
			<td>0.39</td>
			<td>0.18</td>
			<td>0.31</td>
			<td>0.18</td>
		</tr>
		<tr>
			<td>&#964;1 (s)</td>
			<td>5.99</td>
			<td>3.50</td>
			<td>2.46</td>
			<td>2.71</td>
			<td>69.34</td>
			<td>2.36</td>
			<td>14.39</td>
			<td>26.95</td>
		</tr>
		<tr>
			<td>&#964;2 (s)</td>
			<td>96.28</td>
			<td>124.98</td>
			<td>123.87</td>
			<td>88.01</td>
			<td>2.34</td>
			<td>63.35</td>
			<td>83.14</td>
			<td>45.88</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>1.00</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>G0 (Pa)</td>
			<td>6899</td>
			<td>6827</td>
			<td>6825</td>
			<td>6751</td>
			<td>6710</td>
			<td>6771</td>
			<td>6797</td>
			<td>67</td>
		</tr>
		<tr>
			<td>G&#8734; (Pa)</td>
			<td>5967</td>
			<td>6286</td>
			<td>6160</td>
			<td>6197</td>
			<td>6144</td>
			<td>6113</td>
			<td>6145</td>
			<td>105</td>
		</tr>
	</tbody>
</table>
Table 3:&#160;Viscoelastic parameters estimated from indentation tests for the two gelatin phantoms. Each phantom was tested six times.

Watch this Scientific Journal Video about Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography at JoVE.com

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

viscoelastic-characterization-soft-tissue-mimicking-gelatin-phantoms

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.0K Views.  Shanghai Jiao Tong University. This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Video: Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...